T lymphocytes recognize specific antigens through interaction of the T cell receptor (TCR) with short peptides presented by major histocompatibility complex (MHC) class I or II molecules. For initial activation and clonal expansion, naïve T cells are dependent on professional antigen-presenting cells (APCs) that provide additional co-stimulatory signals. TCR activation in the absence of co-stimulation can result in unresponsiveness and clonal anergy. To bypass immunization, different approaches for the derivation of cytotoxic effector cells with grafted recognition specificity have been developed. Chimeric antigen receptors (CARs) have been constructed that consist of binding domains derived from natural ligands or antibodies specific for cell-surface antigens, genetically fused to effector molecules such as the TCR alpha and beta chains, or components of the TCR-associated CD3 complex. Upon antigen binding, such chimeric antigen receptors link to endogenous signaling pathways in the effector cell and generate activating signals similar to those initiated by the TCR complex. Since the first reports on chimeric antigen receptors, this concept has steadily been refined and the molecular design of chimeric receptors has been optimized (for a review see Uherek et al., 2001). Aided by advances in recombinant antibody technology, chimeric antigen receptors targeted to a wide variety of antigens on the surface of cancer cells and of cells infected by human immunodeficiency virus (HIV) have been generated (for a review see Uherek et al., 2001).
US 2007/0031438 A1 describes a CAR comprising a binding domain of an antibody against prostate specific membrane antigen (PSMA), a modified CD8 hinge in which at least one of the cysteine residues has been mutated and a zeta signaling domain of the T cell receptor. In particular, US 2007/0031438 A1 uses a human CD8 hinge region with amino acid positions 135 to 180 (according to the amino acid numbering of Swissprot P01732), wherein the cysteine in position 164 is substituted with alanine.
Fitzer-Attas et al. (1998) describe a CAR comprising a non modified CD8 hinge region with amino acid positions 116 to 208 (according to the amino acid numbering of Swissprot P01732), which comprises three cysteine residues at positions 164, 181 and 206. The chimeric receptor furthermore uses kinase domains as effector domain.
WO 2008/045437 A2 describes CARs comprising as an extracellular binding portion, a single chain antibody portion that binds to EGFRvIII, a transmembrane portion derived from human CD8 alpha or CD28, and an intracellular signaling portion derived from human CD3 zeta. In particular, WO 2008/045437 A2 describes chimeric T cell receptor proteins with a non modified CD8 hinge region with amino acid positions 135 to 205, 135 to 203 or 135 to 182 (according to the amino acid numbering of Swissprot P01732), each comprising cysteine residues in positions 164 and 181.
WO 95/30014 A1 describes a CAR comprising an antigen binding domain derivable from a monoclonal antibody directed against a suitable antigen on a tumor cell (such as scFv(FRP5)), a hinge region comprising from 40 to 200 amino acids and a functional zeta chain derivable from the T cell antigen receptor. In particular, the CAR of WO 95/30014 A1 uses the non modified murine CD8 hinge region with amino acid positions 132 to 191 (according to the amino acid numbering of Swissprot P01731), comprising a cysteine residue in position 178.
US 2008/0260738 A1 describes antibody fusion proteins comprising at least two Fc monomers and at least one linker, wherein a modified CD8 hinge region is used for linking the two Fc monomers. In particular, US 2008/0260738 A1 uses modified CD8 hinge regions with amino acid positions 131 to 170 or 136 to 169 (according to the amino acid numbering of Swissprot P01732), wherein the cysteine in position 164 is substituted with serine.
The present invention aims to provide optimized chimeric antigen receptors which allow more efficient surface expression and high functionality in lymphocytes.
It is a further objective of the present invention to provide means and methods for generating antigen-specific effector cells as well as means and methods for the use in adoptive, target-cell specific immunotherapy and for treatment of cancer.